First introduced in 1997, over 125 million hectares of licensed genetically modified (GM) crops were grown across the globe in 2008 (www.isaaa.org). The primary method used to develop GM crops is dependent on using the soil inhabiting bacteria Agrobacterium tumefaciens to transfer a select gene(s) of interest (e.g. a gene conferring resistance to drought) into a specific plant (e.g. wheat).
Termed Agrobacterium tumefaciens mediated transformation (ATMT), the process of generating GM plants using Agrobacterium tumefaciens is comprehensively patented by the agri-biotech industry for the majority of the globe's commodity crops (Nottenburg C, Rodriguez C R (eds) (2007) Agrobacterium-mediated gene transfer: A lawyer's perspective. Springer, New York). So, it is of considerable importance both academically and commercially to identify and develop other viable non-Agrobacterium bacteria that are capable of mediating cellular transformation. Brooetharts et al. (Nature. 2005 Feb. 10; 433(7026):629-33) described the potential of three non-Agrobacterium strains to genetically transform plant (rice, tobacco and the model plant species Arabidopsis) tissue. However, the transformation efficiency of these “Transbacter” strains was poor relative to standard Agrobacterium-mediated transformation. For example; the best performing Transbacter strain (Sinorhizobium meliloti) transformed Arabidopsis at a rate representing 5-10% of Agrobacterium-mediated transformation and while Brooetharts et al. report transformation frequency in tobacco for the same strain at 28%-36%, this data only represents the recovery of un-rooted shoots. The issue of using bacteria strains such as Transbacter (and related Rhizobia spp.) is further compounded by the necessity for strain-specific optimisations as reported in Brooetharts et al. and Wendt et al. (Transgenic Research, DOI: 10.1007/s11248-010-9423-4Online First™) which complicates transformation protocols relative to the conventional Agrobacterium-mediated transformation protocols that are widely practised. The low transformation efficiencies of rhizobia species are further demonstrated in Wendt et al., where the frequency (calculated as % of shoots equipped with root systems with the ability to grow in rooting media supplemented with 25 μg/ml hygromycin) of transforming potato with the rhizobia strains was calculated at 4.72, 5.85 and 1.86% for S. meliloti, R. sp. NGR234 and M. loti respectively. This differs significantly with an average transformation frequency of 47.6% for the A. tumefaciens control treatment.
International Patent Application No: PCT/US2007/069053 describes the use of a number of non-Agrobacterium strains to genetically transform plant tissue, including transformation of soy using Sinorhizobium freddi SF4404 which achieved a transformation efficiency of 0.04% and transformation of corn using Sinorhizobium freddi SF4404 and Sinorhizobium freddi SF542C which achieved a transformation efficiency of 5.17% and 1.61%, respectively. These contrast with available literature which indicates that ATMT of soybean and corn can achieve transformation efficiencies of up to 18% for soybean (Dang et al., Plant Science, 2007, 173; 381-389) and 22% for corn (Reyes et al., Plant Physiology, 2010, 153:624-631). This indicates that the Sinorhizobium mediated transformation of corn and soy would have a relative transformation efficiency (relative to ATMT) of about 7% to 30%. Similarly, for canola, Patent Application No: PCT/US2007/069053 reports a transformation efficiency of up to 1.33% (RL2370G), which is 18-fold less efficient than reported transformation efficiencies (up to 25%) for ATMT (Cardoza and Stewart, Plant Cell Reports, 2003, 21; 599-604). Thus, the literature clearly indicates that transformation efficiencies achieved using non-Agrobacterium mediated transformation are poor relative to Agrobacterium-mediated transformation, across a number of plants species.
It is an object of the present invention to overcome at least one of the above problems.